Proceedings of the 2nd International Workshop on Materials Analysis and Processing in Magnetic Fields,
(Grenoble, France, March 2006), edited by J. Torbet, S. Rivoirard, and E. Beaugnon (CNRS Grenoble, 2007) pp. 67 – 70.
EFFECTS OF HIGH MAGNETIC FIELDS ON IN VITRO
TRANSCRIPTION
Marianna Worczak 1, Kimberly Wadelton 1, James Ch. Davis 1,
Anna-Lisa Paul 2, Robert J. Ferl 2, and Mark W. Meisel 1*
1
Department of Physics and the National High Magnetic Field Laboratory (NHFML),
University of Florida, Gainesville, FL 32611-8440, USA.
*corresponding author: meisel@phys.ufl.edu
2
Department of Horticultural Sciences and the Biotechnology Program,
University of Florida, Gainesville, FL 32611-0690, USA.
Abstract : Commercially available T7 and SP6 RNA polymerase kits were used to study the effects of
high magnetic fields on in vitro transcription preformed in the presence of magnetic fields ranging up to
9 Tesla. Although full-length transcripts were generated under all test conditions, there was a reduction in
the efficiency of transcription with increasing magnetic field strength.
Keywords : T7 RNA polymerase, SP6 RNA polymerase, biological effects of high magnetic fields
substantial distortions, are hypothesized
to have an effect on enzymatic function.
To test the hypothesis, experiments
employing commercially available T7
and SP6 RNA polymerase in vitro
transcription kits were preformed in the
presence of magnetic fields ranging up to
9 Tesla.
1. INTRODUCTION
Work involving exposure of Arabidopsis
thaliana to high magnetic fields noted an
induction of a gene reporter driven by the
alcohol dehydrogenase promoter [Paul et
al., 2005]. This observation suggested
that a high magnetic field can impact
biological processes at the level of gene
expression. The work presented here
utilizes in vitro transcription to reduce the
complexity of the process. The working
hypothesis fueling our experiments was
that strong magnetic fields acting on the
diamagnetic anisotropies [Worcester,
1978; Pauling, 1979; Torbet, 2005]
already present in the RNA polymerases
might lead to an alteration in the three
dimensional shape of the proteins. These
alterations, whether partial alignments or
2. RNA POLYMERASES
Over the past several years, T7 RNA
polymerase has received considerable
attention due to its role in transcription
[Ellenberger, 1999]. In particular, x-ray
studies have provided the structure
[Jeruzalmi & Steitz, 1998] and insight
into the conformation of the initiation
complex (IC) and elongated complex
(EC) [Cheetham & Steitz, 1999;
Cheetham et al., 1999; Tahirov et al.,
67
Proceedings of the 2nd International Workshop on Materials Analysis and Processing in Magnetic Fields,
(Grenoble, France, March 2006), edited by J. Torbet, S. Rivoirard, and E. Beaugnon (CNRS Grenoble, 2007) pp. 67 – 70.
2002; Yin & Steitz, 2002], but the
dynamical details are not presently
resolved [Thesis et al., 2004; Guo et al.,
2005].
Nevertheless, T7 RNA
polymerase is an excellent model system
for testing our hypothesis.
tube and subsequently deposited into
liquid nitrogen to terminate the reaction.
The sample remained in the liquid
nitrogen until all time points were
completed. The RQ1-RNase-free DNase
was then added in a concentration of
1μl/μg of template to remove the DNA
template and ensure that the reaction was
indeed terminated. Reactions were then
stored at 20ºC until analyzed.
On the other hand, SP6 RNA polymerase
is less well characterized at the structural
level, but the parallels in residue
sequence and functional activity suggest
the SP6 structure is similar to that known
for T7 [Kotani et al., 1987].
Standard agarose gel electrophoresis was
used to determine the lengths and
quantities of the transcripts. Samples
were run in denaturing agarose gels
containing formaldehyde and ethidium
bromide. A volume of 4μl of each
reaction mixture was combined with 8μl
of RNA loading buffer. The samples
were then incubated at 55°C for
10 minutes to allow for denaturation.
Next, a total volume of 9µl of each
sample was loaded into the gel, which
was run at a constant voltage of 100 V for
25 minutes. Gel images were obtained by
a Kodak Gel Logic 440 Imaging System
and Kodak 1D 3.6 software.
Final
analysis was performed with BioRad
Quantity One gel imaging software.
3. EXPERIMENTAL DETAILS
Reactions were performed to evaluate the
production of transcripts generated from
the control template (pGEM DNA)
provided in the Ribomax T7 and SP6
Express In Vitro Transcription Kits
[Promega, 2005]. The template was
chosen
to
ensure
the
simplest
experimental conditions feasible for
magnet runs. Reactions included controls
at ambient temperatures for T7 and at
37°C for SP6. For a given set of
reactions, a mastermix of reaction buffer,
nuclease-free water, and control template
was prepared.
This solution was
subsequently split into the necessary
number of aliquots, and once divided, the
individual samples were frozen at 20ºC
and stored until reactions were
completed.
Control reactions were
performed in the bore of the magnet prior
to generation of a magnetic field. For
each magnetic field strength, the enzyme
was carefully placed atop the frozen
mixture, and then lowering the sample
into the sample vesicle initiated the
reactions. A 10μl aliquot of the reaction
was removed at each time point (1, 5, 10
and 20 minutes). Each aliquot was
quickly placed into a separate sample
A standard Oxford Instruments 9 Tesla
NMR superconducting solenoid with a
89 mm room temperature bore was used
at low fields. The sample tubes were
held at the center of the magnets by
means of a simple plastic bucket
suspended by string.
For the SP6
samples, a 37°C environment was
provided by thermally-regulated hot
water circulating though copper tubing.
A standard thermocouple was used to
monitor the temperature. For all runs, the
1ml sample tubes were attached to a rigid
rod and carefully placed into the bucket.
When a time course was complete, the
68
Proceedings of the 2nd International Workshop on Materials Analysis and Processing in Magnetic Fields,
(Grenoble, France, March 2006), edited by J. Torbet, S. Rivoirard, and E. Beaugnon (CNRS Grenoble, 2007) pp. 67 – 70.
tube was quickly removed and then
replaced.
4. RESULTS
The T7 RNA polymerase results are
shown in Figures 1 and 2, while Figure 3
shows the results from SP6 RNA
polymerase.
Figure 3. Agarose gel electrophoresis results
using SP6 RNA polymerase are shown for runs in
zero field (controls), 4.5 Tesla, and 9 Tesla. The
reactions were halted at time points of 1, 5, 10
and 20 minutes. The control template used for the
reactions produces a band 1.8 kb.
5. DISCUSSION
Figure 1. Agarose gel electrophoresis results
using T7 RNA polymerase are shown for runs in
zero field (controls), 4.5 Tesla, and 9 Tesla. The
reactions were halted at time points of 1, 5, 10
and 20 minutes. The control template used for the
reactions produces bands at 1.1 kb (bottom band)
and 2.3 kb (top band).
I(t) / I(t = 1 min)
20
The results suggest that the RNA
production using T7 RNA polymerase
was reduced in the presence of the
magnetic fields.
For SP6 RNA
polymerase, the data suggest a reduced
production rate in 9 Tesla. Although the
differences in the production rates are not
distinguishable at the level of “two
sigma” (standard deviations), see Figure
2, the observed trends are worthy of
additional, systematic studies exploring a
greater range of magnetic field and time.
Controls
4.5 Tesla
9.0 Tesla
15
10
5
0
0
5
10
15
20
25
It is noteworthy that the present
experiments were initiated for the
purpose of establishing a low field
baseline for studies proposed between 15
and 25 Tesla. Our expectations were
based upon our earlier work [Paul et al.,
2005] and on studies of the gene
expression profiles of yeast in magnetic
fields up to 14 Tesla [Ikehata et al.,
2003].
30
Time (minutes)
Figure 2. The Intensity of the bands in Fig. 1 (T7
RNA polymerase) are shown as a function of
time. The data have been normalized to the
results at t = 1 min., and the background has been
subtracted. The straight lines are linear fits to the
data, and the uncertainty bars at 22.5 min. indicate
the standard deviations.
At this time, our working hypothesis
remains
a
plausible
possibility.
Additional experiments are required to
69
Proceedings of the 2nd International Workshop on Materials Analysis and Processing in Magnetic Fields,
(Grenoble, France, March 2006), edited by J. Torbet, S. Rivoirard, and E. Beaugnon (CNRS Grenoble, 2007) pp. 67 – 70.
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4101-4113.
Kotani, H. et al., 1987. Nucleotide sequence and
expression of the cloned gene of bacteriophage SP6
RNA polymerase. Nucleic Acids Res., Vol. 15, pp
2653-2664.
Paul, A.-L. et al., 2005. Strong Magnetic Field Induced
Changes of Gene Expression in Arabidopsis.
Materials Processing in Magnetic Fields:
Proceedings of the International Workshop on
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Pauling, L., 1978. Diamagnetic Anisotropy of the
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Technical
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of T7 RNA Polymerase Suggests a New Twist.
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test the hypothesis and to explore the
different effects that magnetic fields have
on the T7 and SP6 RNA polymerase
reactions.
ACKNOWLEDGEMENTS
This work was partially supported by the NHMFL
Summer 2005 REU Program at the University of
Florida in Gainesville, NSF DMR-0305371
(MWM), and NASA grant NNA04CC61 (ALP
and RJF).
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